The present application relates to the field of biotechnology, and in particular, to fields involving the study and use of membrane-associated proteins.
All living organisms are composed of cells, from single celled organisms such as bacteria, to the complex cellular architecture of humans. The cells include multifaceted, chemically driven systems, such as, for example, communication networks that control a cell's response to external stimulus. Signal transduction pathways involve protein ‘teams’ that work in concert to execute desired pathway instructions, such as, for example, gene regulation, cell growth, movement, and hormone release.
Cell membranes are bilayers of lipid molecules that define the boundary between, and serve as selective barriers between, the inside and outside of all cells and between the inside and outside of cellular compartments (organelles). Similar membranes also define the boundary between the inside and outside of some viruses. A wide variety of proteins are embedded in or on, or associated with, the cell membrane, thereby creating a highly specialized environment. It is widely accepted that the membrane environment, including the proteins and assemblies of proteins that naturally occur in and on the membrane, is essential for normal biological function. For example, a significant portion of these membrane proteins are responsible for the process of transmembrane signaling, which conveys information across the membrane, frequently, although not exclusively, from the outside of the cell to the inside. The membrane can be likened to a two-dimensional fluid sheet, which serves as the natural template for the assembly of signal transduction elements. The association of these proteins with the membrane in essence restricts their motion to two dimensions rather than three, which promotes interactions between proteins that are necessary for proper assembly and function.
Typically, transmembrane signaling proteins are the transducers of the initial stimuli that set cellular pathways in motion. The signal transduction pathways in which the transmembrane signaling events are a part, are critical for generating responses to broad range of external stimuli that are generally recognized to be generated either by the organism itself (hormones, growth factors, other cells) or from foreign entities (foreign cells or cells recognized as foreign, viruses, bacteria, other pathogens and pathogenic materials, and allergens). Transmembrane signaling and signal transduction pathways are also indispensable for communication among cells in multicellular organisms. Consequently, almost all processes critical to the growth and function of multicellular organisms depend on transmembrane signaling. When these communication networks fail to execute an instruction, or when signaling becomes deregulated, diseases result, such as, for example, cancer, diabetes, and obesity. To illustrate the crucial role of cell signaling in disease, it has been reported that greater than 60% of all drugs, including drugs available in the marketplace and drugs that have been selected for market, target proteins involved in signal transduction pathways. With an estimated annual spending on early stage drug screening in excess of one billion dollars, there is a great need for innovations that improve the efficiency and accuracy of such screening assays.
“Transmembrane receptors” are key protein elements in the process of signal transduction. The receptors often span the membrane bilayer one or more times in order to convey information across it during the process of transmembrane signaling. It is widely known that membrane receptors interact with one another by clustering together in the membrane to form dimers, trimers, or more generally oligomers, and that the process of clustering and/or the formation of multimers is an integral part of the transmembrane signaling process. Dimers, are often generated through the association of two identical protein molecules to form homodimers, but heterodimers can form in other instances, through the specific association of two different receptors (See, e.g., Martin and Wesche, 2002; Bazan-Socha et al. 2005; Penuel et al., 2001). More generally hetero-oligomeric complexes form to orchestrate the transmembrane signaling. (See, e.g., Alarcon et al., 2003). Also, additional proteins involved with the process of transmembrane signaling have been reported to associate with the inner leaflet of the membrane through specific interactions with the receptor and/or the membrane itself. (See, e.g., Pawson and Nash, 2003). These too are part of the process of signal transduction.
Genome sequencing projects have produced a wealth of information that have brought about significant advances in descriptive cellular and molecular biology, including the establishment of familial and evolutionary classifications of a multitude of transmembrane receptors. (See, e.g., Ben-Shlomo et al., 2003). These works, along with the continuing efforts to determine the structures and functions of transmembrane receptors, have, altogether, led to the identification of unifying principles in the processes of transmembrane signaling, principles that are inextricably associated the special properties of the cell membrane.
Significant resources and attention have been devoted to the study of membrane-associated proteins; however, membrane samples of the proteins that are used in such biochemical experiments are frequently isolated from cells expressing the receptor at elevated levels, which can result in complex and heterogeneous samples. Also, receptor reconstitution is labor-intensive, and the conditions that maintain a high level of activity while also preserving the vectoral and lateral organization required for function can be difficult to find. Notably, it is the very association of receptors with membranes that invariably requires the use of detergent for the purification of receptors, which leads to well-known difficulties, including low yield and the disruption of critical protein-protein interactions. Low yields are typical and represent a major impediment to widespread use of such receptors in cell-free assay systems. Also, the solubilizing activity of detergents, which is the basis of their usefulness in other applications, such as membrane protein purification, represents a significant disadvantage in functional assays where protein-protein interactions are necessary. In this setting, detergents disrupt necessary interactions between the receptors in the membrane, as well as the interactions between receptors and receptor-associated proteins, and protein-protein interactions in general. While formulations of detergent compatible with functional activity can sometimes be achieved, these are identified only by time-consuming and case-specific methods, and the level of activity usually achieved often remains less than satisfactory.
To overcome these difficulties, researchers have attempted to identify key regions of the membrane-associated proteins that can be cloned out for study in vitro. Some of these receptor fragments support activity and have been commercialized for the study of pair-wise interactions, such as, for example, interactions between a protein domain that possesses enzymatic activity and a substrate. Much information has been lost in these situations, however, as signaling proteins are studied in environments that differ significantly from their natural, cellular environments.
It is apparent from the above that there is a continuing need for advancements in the relevant field, including new methods and materials for restoring function to membrane-associated proteins outside their natural, cellular environment. The present application addresses this need.